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Food Safety and Toxicology Presentation on Toxicants Generated During

Thermal Processing of Food (Acrylamide)

Presented By, Basavaraj Gani Sadashivappa

INRODUCTION

Acryl amide is a thermally induced toxicant present in different

processed foods in varying amount. Due to its detrimental effect on human health, it has become a major concern in public health and food safety. This presentation aims to review and focuses on the mechanisms of acryl amide formation, the effects of different processing parameters such as pH, temperature, time, types and the amount of raw materials, its toxicity level, and its detection methods in complex food systems. Toxicity levels of acryl amide have been found to be neurotoxin and carcinogenic. Food safety authorities including Codex Alimentarius Commission are in the process of reviewing their standards to fix the limit of acryl amide in processed foods.

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• Acryl amide is a highly reactive unsaturated amide.• Chemical formula is C3H5NO and named as prop-2-enamide

in IUPAC system of nomenclature. • It is a white odourless crystalline solid, highly soluble in

water, ethanol, ether and chloroform.• Non-thermal decomposition of acryl amide produces

ammonia whereas thermal decomposition produces carbon monoxide, carbon dioxide, and oxides of nitrogen

Characteristics of acrylamide

• Parameters Values• Structure:

• IUPAC name: prop-2-enamide• Molecular formula: C3H5NO (CH2CHCONH2)• Molar mass: 71.08 g mol−1• Density: 1.13 g/cm3• Melting point: 84.5 °C• Boiling point: 125 0C• Solubility in water: 2.04 kg/L (at 25 °C)• Flash point: 138 °C

MECHANISM OF ACRYLAMIDE FORMATION

• The main ingredients that are responsible for acryl amide formation in foods are carbohydrates especially reducing sugars and asparagine.

• Maillard reaction has been considered as the major reaction pathways for acryl amide formation while processing of food.

• Three major hypotheses are proposed to describe the mechanism of formation of acryl amide in foods.

1. The first mechanism explains the direct formation of acryl amide from amino acids such as alanine, asparagine and glutamine and from methionine

2. The second mechanism involves the formation of acrylamide via the formation of acrolein

3. The most explained pathway for acrylamide formation is via Maillard reaction between reducing sugars and the amino acid asparagine.

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• High temperature cooking such as frying, roasting, or baking is most likely to cause acrylamide formation.. Acrylamide is found in significant amount in plant based foods such as potato products, grain products or coffee.

• The amount of acrylamide found in dairy, meat, and fish products is negligible.

• Generally acrylamide is more likely to accumulate when cooking is done for longer periods or at higher temperatures. It is generally formed at temperatures higher than 120ᵒC and increases with increasing frying and baking temperatures.

A pathway of Acrylamide formation from amino acid and reducing sugar

Asparagine + Glucose (HEAT) Schiff’s base (DECARBOXYLATION) Amadori re-arrangement (B-ELIMINATION) Acryl amide

Alternative routes for formation of acrylamide

Fatty Acids Glycerol

Aspartic acid CarnosineB-Alanine

Acrolein Acrylic Acid

SerineCysteine

Pyruvic acid

Acryl Amide

Sources in Human Nutrition

Food commodities Maximum acryl amide content in PPM

In EU In US FDA

Breads 12-3200 <10-364

Crackers and biscuits <30-2000 26-504

Toasted cereals <30-3200 52-266

French fries 85-1104 20-1325

Vegetables 10-<50 <10-70

Meats <30-64 <10-116

Candy and desserts <20-110 <10-909

Coffee and tea 170-700 175-351

Dairy products 10-100 <10-43

Infant foods 40-120 <10-130

Absorption of Acryl Amide• Absorption of Acrylamide has traditionally been researched using the oral and

intraperitoneal modes of introduction into the body with relatively little research into inhalation exposure. Absorption is closely linked with the mode of environmental exposure to the human body. Acryl amide's half-life in the open air is relatively short in comparison to its other modes of transportation.

• Studies conducted involving oral exposures in rats have yielded results showing that Acrylamide is readily absorbed into the GI tract .

• A comparison of oral vs. intraperitoneal exposure was conducted showing that systemic absorption following oral exposure was less versus that of intraperitoneal exposure.

• Oral exposure has been the primary focus of Acryl amide's main entry into the human and thus a heavy focus of tissue research into its toxicology upon GI tract tissues has taken place.

• Another tissue susceptible to Acrylamide absorption is in nervous tissue of the peripheral nervous system. High uptake levels of Acrylamide in the motor nerve terminus and optic nerve are two areas, which have been shown to result in degeneration of the distal axon terminal. Essentially, this process of dying back allows more acrylamide to enter the cell causing further intoxication. Earliest neuromuscular changes can be seen in the pacinian corpuscles followed by the muscle spindles and finally the motor nerve terminus.

• In the optic nerve, mid diameter axons are most prone to acrylamide absorption in the optic tract allowing for neuronal degeneration in tested primates.

Continue..• Acrylamide metabolism can have 2 possible pathways in route to the excretion of

the compound. Pathway 1 involves utilizing the Cytochrome P450 metabolic enzyme to transform Acrylamide to an easier excrete able Gylcidamide. Pathway 2 involves the conjugation of Acrylamide to Glutathione (GST) there by forming N-acetyl-S- (3-amino-3-oxopropyl) Cysteine as the final product.

• Differentiation between pathways 1 versus pathway 2 is dependant on the species absorption of Acrylamide. Area specific metabolism in rats has shown that the liver, kidney, brain, and erythrocyte GST has a significant higher affinity for binding Acrylamide with the liver being 3 times more prone to Acrylamide binding versus the other mentioned target organs. Solubility of the compound and its method of absorption is highly dependant on the GST pathway for conjugation to a water-soluble product. The conversion by GST to Gylcidamide results in the chemical creation of a radical epoxide, which can express toxicity independently from other neuropathies associated with Acrylamide.

• Studies have been conduct in rats, which indicate that some species of rats may in fact inhibit GST conjugation thus allowing for increased amounts of the toxic product Gylcidamide.

Toxicity• Human exposure to acrylamide primarily comes from dermal contact with solid

monomer and inhalation of dust and vapour in the occupational setting• Humans may be exposed to acrylamide through the ingestion of drinking water

that is contaminated with acrylamide or the intake of acrylamide from food.• Major health effects of acrylamide are skin irritation such as redness and peeling

of the skin of palms and neuropathy regarding the central nervous system and the peripheral nervous system. Acute and sub-acute intoxication with a large dose by ingestion by water contaminated with acrylamide can cause severe symptoms like:

1. Acute toxicity : Acrylamide is a skin and respiratory tract irritant in humans. Reported oral LD50 values are in the range of 159 mg/kg to 300 mg/kg body weight in rats the central nervous system and polyneuropathy may appear later

2. Chronic Toxicity : Acrylamide is a human neuro-toxicant. Adverse effects in rats administered small amounts of acrylamide include general systemic toxicity and haematological changes. Acrylamide is also a neuro-toxicant to animals.

a. In rats, repeated oral administration of acrylamide at doses of 20 mg/kg bw/day and above produced peripheral neuropathy, At 5 mg/kg bw/day in a 90-day study in rats, peripheral lesions occurred and slight changes in peripheral nerve tissue occurred.

b. In monkeys, clinical signs of peripheral neuropathy occurred at doses of 10 mg/kg bw/day for up to 12 weeks.

c. Carcinogenicity : Although inadequate evidence is available from human studies, several laboratory animal studies have shown that acrylamide causes a variety of tumours in rats and mice. Acrylamide has been classified by the U.S. EPA as a B2, a probable human carcinogen, by IARC as a 2B, a possible human carcinogen, and by ACGIH as an A3, confirmed animal carcinogen with unknown relevance to human.

d. Genotoxicity : Acrylamide causes chromosomal aberrations, dominant lethality, chromatic exchanges and unscheduled DNA synthesis in various in vitro and in vivo systems. When administered at a level of 500 ppm in the diet for 3 weeks in mice, acrylamide caused a high frequency of chromatic exchanges.

e. Neurotoxicity : Acrylamide is a neurotoxin by either oral (in animals) or inhalation exposure (in humans and in animals). Toxic effects are central and peripheral neuropathy causing drowsiness, hallucinations and distal numbness. Recovery is possible after cessation of exposure. EPA has derived an oral reference dose (RfD) of 0.0002 mg/kg/day for acrylamide, based on adverse nervous system effects in laboratory animals.

Determination of Acryl Amide• Various methods are available to determine the acrylamide content

of food. The most common methods utilize either GC-MS or LC-MS • Water is often used as the extraction solvent. Solid-phase

extraction (SPE) is typically use to prepare samples. SPE phases that have been used for this method include graphitized carbon black, mixed mode anion and cation exchange, and polymeric materials.

• The GC-MS method of determining acrylamide content in food is well established. A water extract of the food is brominated to form 2,3-dibromopropionamide, a derivative of acrylamide with enhanced GC properties.

• The derivative is then analyzed by GC-MS using Methacrylamide as an internal standard . Detection limits for the GC-MS method are typically in the 5–10 µg/kg range

Steps in Acryl amide analysis by GC/MS Method

• Acryl amide extracted with water, test portion homogenized and acidified to pH 4-5

• Addition of carrez 1 and carrez solution

• Extraction with Ethyl acetate hexane (80:20), Filtration over Na2So4

• Clean up with flurosile elution of acrylamide with acetone

• Evaporation residue taken up in ethyl acetate, tri ethylamine added

• Filtration, injection into GC-MS

• Identification and Quantification

FDA Action Plan for Acrylamide in Food

WHO/FAO Recommendations• In June 2002, the World Health Organization (WHO) and the Food

and Agriculture Organization (FAO) convened an expert consultation on acrylamide. The consultation, in which three FDA experts participated, concluded that the presence of acrylamide in food is a major concern, and recommended more research on mechanisms of formation and toxicity. Both the WHO/FAO consultation and the FDA have recommended that people continue to eat a balanced diet rich in fruits and vegetables. The WHO/FAO consultation advised that food should not be cooked excessively, i.e., for too long a time or at too high a temperature, but also advised that it is important to cook all food thoroughly--particularly meat and meat products--to destroy food borne pathogens (bacteria, viruses, etc.) that might be present.

Further actions

• FDA will develop and revise regulatory options as additional knowledge is gained on acrylamide in food. Many of the items in the action plan are geared toward achieving that end.

• FDA will encourage industry to adopt feasible, practical, and safe processes that are successful at reducing acrylamide, if needed.

• FDA will develop and revise consumer messages about dietary choices and cooking methods, as additional knowledge is gained about acrylamide in food. FDA understands the importance of consumer messages that can assist consumers in making informed dietary choices. Any adjustments in consumer messages would consider the totality of the science.